The HERG protein encoded by KCNH2 constitutes the pore forming subunit of IKr, a K+ current that plays a key role in repolarization of the cardiac action potential in the atrium and ventricle. Mutations in KCNH2 predispose patients to ventricular arrhythmias (Long QT syndrome;LQTS), and block of the channel by antiarrhythmic or other drugs is the commonest form of the acquired, or drug-associated, LQTS. This form of proarrhythmia is one of the major limitations for currently used antiarrhythmic drug therapies, and remains incompletely understood. Moreover, an increasing body of evidence indicates that atrial fibrillation (AF), the most commonly-treated cardiac arrhythmia, may also arise as a result of mutations in ion channel genes. Our collaborators at VUMC have identified KCNH2 mutations in patients with AF, and some of these have in fact been previously reported as causes of congenital LQTS. This raises the question-to be addressed here-of the mechanisms underlying individual variability in the clinical response to KCNH2 mutations or to drugs that block IKr. We have previously identified KCR1 as a HERG partner, and more recently, using a C. elegans model, we have identified new genes that regulate HERG membrane expression (ARHGAP6, GSK-3[unreadable]) through specific domains in the HERG C-terminus. Our central hypothesis is that HERG C-terminal sequence variants, and proteins that regulate HERG activity, impact the function of HERG in a chamber- specific manner, and thereby modulate the channel's contribution to normal and abnormal action potentials, and to its response to drug exposure. In Specific Aim 1, we will compare the effects of the AF-associated HERG C-terminal mutations to previously-identified, neighboring or overlapping LQTS mutations using a combination of electrophysiology, biochemistry and computational modeling. In Specific Aim 2, we will compare the spatial distribution of HERG modulators KCR1, ARHGAP6 and GSK-3[unreadable], in atrium vs. ventricle. Using heterologous expression and quantitative modeling, we will explore the impact of these molecules on repolarization in the atrium and ventricle. We will similarly assess HERG modulators previously reported in the literature, as well as new modulators identified through GST-pulldown assays from mouse cardiac myocytes. In Specific Aim 3, we will finalize a preliminary NMR solution structure of the distal HERG C-terminus, with a view to better understanding how the stability of structured domains within the distal C-terminus is impacted by AF/LQTS-linked mutations. An improved understanding of how these factors influence the function and chamber-specific distribution of HERG will clarify how sequence variants may put AF patients at risk for therapeutic failure or for proarrhythmia during drug treatment. These results may also identify new targets for atrial-specific antiarrhythmic intervention. PUBLIC HEALTH RELEVANCE: Cardiac arrhythmias continue to be a major public health problem: 2-5 million American suffer from atrial fibrillation and 15% succumb to sudden cardiac death from ventricular arrhythmias;this proposal will consider common and divergent aspects of both atrial and ventricular arrhythmias. It will contribute to the identification of patient risk factors predisposing to arrhythmia development, as well as identifying risk associated with drug therapy commonly administered for cardiac and non-cardiac conditions. Completion of the project will enhance our understanding of the genetic, molecular and physiological mechanisms underlying arrhythmia disorders.